Adult Erythroid Cells Research Articles

Human embryonic hemopoiesis: Control mechanisms underlying progenitor differentiation in vitro

In order to investigate differences in control mechanisms between embryonic and adult hemopoiesis, we have studied the sensitivity of human embryonic progenitors (5–8 weeks postconception) to either positive (erythropoietin (Ep), granulocyte-macrophage colony-stimulating factor (GM-CSF) and insulin-like growth factor 1 (IGF-1)) or negative (tumor necrosis factor (TNF) and interferon-γ (IFN-γ)) in vitro regulators of adult hemopoietic differentiation. Growth stimulators were analyzed under serum-deprived conditions whereas growth inhibitors were investigated in serum-supplemented culture. Formation of granulocyte-macrophage colonies from embryonic progenitors was induced by GM-CSF but inhibited by TNF and IFN-γ. Early erythroid progenitors resemble adult erythroid burst-forming cells (BFU-E) in their sensitivity to Ep and TNF but differ in their lack of response to GM-CSF or other adult sources of burst-promoting activity, and absence of inhibition by IFN-γ. IGF-1 promoted erythroid burst formation in the absence of insulin, but did not have Ep-like activity. These data indicate that embryonic and adult erythroid progenitors differ at least in terms of in vitro sensitivity to GM-CSF and IFN-γ and suggest that different cellular response to control signals may underlie the differences observed in vivo between embryonic and adult hemopoiesis.

Upstream Gℽ-Globin and Downstream β-Globin Sequences Required for Stage-Specific Expression in Transgenic Mice

The human G gamma-globin and beta-globin genes are expressed in erythroid cells at different stages of human development, and previous studies have shown that the two cloned genes are also expressed in a differential stage-specific manner in transgenic mice. The G gamma-globin gene is expressed only in murine embryonic erythroid cells, while the beta-globin gene is active only at the fetal and adult stages. In this study, we analyzed transgenic mice carrying a series of hybrid genes in which different upstream, intragenic, or downstream sequences were contributed by the beta-globin or G gamma-globin gene. We found that hybrid 5'G gamma/3'beta globin genes containing G gamma-globin sequences upstream from the initiation codon were expressed in embryonic erythroid cells at levels similar to those of an intact G gamma-globin transgene. In contrast, beta-globin upstream sequences were insufficient for expression of 5'beta/3'G gamma hybrid globin genes or a beta-globin-metallothionein fusion gene in adult erythroid cells. However, beta-globin downstream sequences, including 212 base pairs of exon III and 1,900 base pairs of 3'-flanking DNA, were able to activate a 5'G gamma/3'beta hybrid globin gene in fetal and adult erythroid cells. These experiments suggest that positive regulatory elements upstream from the G gamma-globin and downstream from the beta-globin gene are involved in the differential expression of the two genes during development.

Upstream G gamma-globin and downstream beta-globin sequences required for stage-specific expression in transgenic mice.

The human G gamma-globin and beta-globin genes are expressed in erythroid cells at different stages of human development, and previous studies have shown that the two cloned genes are also expressed in a differential stage-specific manner in transgenic mice. The G gamma-globin gene is expressed only in murine embryonic erythroid cells, while the beta-globin gene is active only at the fetal and adult stages. In this study, we analyzed transgenic mice carrying a series of hybrid genes in which different upstream, intragenic, or downstream sequences were contributed by the beta-globin or G gamma-globin gene. We found that hybrid 5'G gamma/3'beta globin genes containing G gamma-globin sequences upstream from the initiation codon were expressed in embryonic erythroid cells at levels similar to those of an intact G gamma-globin transgene. In contrast, beta-globin upstream sequences were insufficient for expression of 5'beta/3'G gamma hybrid globin genes or a beta-globin-metallothionein fusion gene in adult erythroid cells. However, beta-globin downstream sequences, including 212 base pairs of exon III and 1,900 base pairs of 3'-flanking DNA, were able to activate a 5'G gamma/3'beta hybrid globin gene in fetal and adult erythroid cells. These experiments suggest that positive regulatory elements upstream from the G gamma-globin and downstream from the beta-globin gene are involved in the differential expression of the two genes during development.

A 3' enhancer contributes to the stage-specific expression of the human beta-globin gene.

The human beta-globin and G gamma-globin genes are expressed at different stages of human development and also show distinct temporal patterns of expression when transferred into the mouse germ line. In transgenic mice, the beta-globin gene is expressed only in fetal and adult erythroid cells, whereas the G gamma-globin gene is active only in embryonic erythroid cells. Previous experiments suggested that beta-globin 3' sequences were important for expression in mouse fetal and adult erythroid cells, and in this paper we directly demonstrate the presence of an enhancer in the 3'-flanking region of the gene. First, deletion of sequences between 605 and 895 bp, 3' to the poly(A) site, results in a 10-fold reduction in the average level of expression of the beta-globin gene in transgenic mouse fetal livers. Second, a DNA fragment including beta-globin 3'-flanking sequences [425-1480 bp from the poly(A) site], in either orientation, activates transcription from the otherwise silent G gamma-globin promoter in the mouse fetal liver; DNA sequences between 150 and 730 bp or between 920 and 1680 bp, 3' to the beta-globin gene, are inactive by this assay. Together, these experiments identify an enhancer, in the region approximately 600-900 bp, 3' to the beta-globin poly(A) site, which contributes to the differential stage-specific expression of the beta-globin and G gamma-globin genes.

Sardinian δβ°-Thalassemia: A Further Example of a C to T Substitution at Position - 196 of the Aγ Globin Gene Promoter

Selective overexpression (50- to 100-fold) in adult erythroid cells of either G gamma or A gamma fetal globin gene is observed in hereditary conditions known as delta beta zero-thalassemia and hereditary persistence of fetal hemoglobin (HPFH). Recently, a C----T change at position -196 of an overexpressed A gamma globin gene from an Italian HPFH was hypothesized, on the basis of indirect evidence, to represent the cause of the functional defect. We now show that the same mutation is present in a different overexpressed A gamma-globin gene from a Sardinian patient with a different syndrome (delta beta zero-thalassemia). The Sardinian A gamma globin gene differs from both the HPFH and the normal A gamma globin gene at nucleotide 1,560 in the noncoding portion of the third exon, where an A is deleted. In addition, the mutant -196 A gamma-globin gene is linked to a normal beta globin gene in HPFH, and to a beta-thalassemic gene (beta 39CAG----TAG) in delta beta zero-thalassemia. These data strengthen the suggestion that -196 mutation is causally linked to the abnormal phenotype and raise the question of whether the same or multiple mutational events are responsible for the appearance of the -196 mutation in different syndromes.

Sardinian delta beta zero-thalassemia: a further example of a C to T substitution at position -196 of the A gamma globin gene promoter

Selective overexpression (50- to 100-fold) in adult erythroid cells of either G gamma or A gamma fetal globin gene is observed in hereditary conditions known as delta beta zero-thalassemia and hereditary persistence of fetal hemoglobin (HPFH). Recently, a C-T change at position -196 of an overexpressed A gamma globin gene from an Italian HPFH was hypothesized, on the basis of indirect evidence, to represent the cause of the functional defect. We now show that the same mutation is present in a different overexpressed A gamma-globin gene from a Sardinian patient with a different syndrome (delta beta zero- thalassemia). The Sardinian A gamma globin gene differs from both the HPFH and the normal A gamma globin gene at nucleotide 1,560 in the noncoding portion of the third exon, where an A is deleted. In addition, the mutant -196 A gamma-globin gene is linked to a normal beta globin gene in HPFH, and to a beta-thalassemic gene (beta 39CAG-TAG) in delta beta zero-thalassemia. These data strengthen the suggestion that -196 mutation is causally linked to the abnormal phenotype and raise the question of whether the same or multiple mutational events are responsible for the appearance of the -196 mutation in different syndromes.

Fine analysis of the active H5 gene chromatin of chicken erythroid cells at different stages of differentiation

We have analyzed the chromatin structure of a region that encompasses 14.4 × 10 3 basepairs of the chicken histone H5 locus in adult erythroid cells at different stages of maturation. Seven of eight major lineage-specific DNase I-hypersensitive sites, some of which show complex substructure, were found in the flanking regions of the gene. The hypersensitivity of some of these sites is modulated during erythrocyte maturation in a way that parallels the transcriptional activity of the gene. DNase I, micrococcal nuclease, and S 1 nuclease recognize the same regions, which differ from those cleaved by S 1 on supercoiled plasmid DNA. This suggests that hypersensitivity of DNA in chromatin reflects a greater accessibility of the DNA rather than its altered conformation. The DNA sequence of some of the DNase I target sites contains repeated motifs, (T-C-C-C) 2, (T-C-C) 2, (T-G-G-G-G) 2, which are found in the hypersensitive sites of other genes. Detailed analysis across sections of the H5 gene and flanking sequences revealed differences in the DNase I sensitivity of the different regions examined. Notably, the first one-third of the gene is more sensitive than the rest. The sequences downstream from the region where most RNA polymerases terminate transcription were found to be the most resistant.

Acetylsalicylic acid antagonizes the bleeding-induced changes in hemoglobin proportions in normal adult rats

Normal adult Sprague-Dawley rats were made anemic by repeated phlebotomy. Ion-exchange chromatography of anemic blood showed newborn like hemoglobin proportions, involving the same six hemoglobin components as is found when newborn and adult blood are compared. However, acetylsalicylic acid intake during anemia failed to demonstrate the changes in hemoglobin proportions, either totally or partially, depending upon the doses. Since acetylsalicylic acid inhibits prostaglandin synthesis, the data suggest that one or more prostaglandins may be involved in the process of reverse switching of hemoglobin in adult rat erythroid cells during erythropoietic stress.

Differences in DNase I sensitivity and methylation within the human beta-globin gene domain and correlation with expression

We have analysed the chromatin features of DNA regions encompassing human epsilon, G gamma, A gamma, delta and beta globin structural genes in fetal and adult erythroid cells on the one hand and adult lymphocytes on the other. Highly purified nuclei from these cells were submitted to DNase I digestion and the kinetic data were obtained from the percentage of residual hybridization of defined regions in Southern blots. Our results, as others have shown by different approaches, indicate that the structural genes of the beta-globin cluster are generally more sensitive to DNase I in the erythroid cells than in non-erythroid cells. Thus a domain of DNase I sensitivity related to the committed state is defined. In addition we show that within this DNase-I-sensitive beta cluster domain, individual genes of the cluster are arranged in subdomains of differential DNase I sensitivity, which correlate with their expression status. Furthermore the differential expression of the two fetal genes in the fetal stage is shown to be directly proportional to the degree of hypomethylation of these genes.

Chromatin structure and developmental expression of the human alpha-globin cluster.

The human alpha-like globins undergo a switch from the embryonic zeta-chain to the alpha-chain early in human development, at approximately the same time as the beta-like globins switch from the embryonic epsilon-to the fetal gamma-chains. We investigated the chromatin structure of the human alpha-globin gene cluster in fetal and adult erythroid cells. Our results indicate that DNase I-hypersensitive sites exist at the 5' ends of the alpha 1- and alpha 2-globin genes as well as at several other sites in the cluster in all erythroid cells examined. In addition, early and late fetal liver erythroid cells and adult bone marrow cells contain hypersensitive sites at the 5' end of the zeta gene, and in a purified population of 130-day-old fetal erythroid cells, the entire zeta-to alpha-globin region is sensitive to DNase I digestion. The presence of features of active chromatin in the zeta-globin region in fetal liver and adult bone marrow cells led us to investigate the transcription of zeta in these cells. By nuclear runoff transcription studies, we showed that initiated polymerases are present on the zeta-globin gene in these normal erythroid cells. Immunofluorescence with anti-zeta-globin antibodies also showed that late fetal liver cells contain zeta-globin. These findings demonstrate that expression of the embryonic zeta-globin continues at a low level in normal cells beyond the embryonic to fetal globin switch.

Chromatin structure and developmental expression of the human alpha-globin cluster.

The human alpha-like globins undergo a switch from the embryonic zeta-chain to the alpha-chain early in human development, at approximately the same time as the beta-like globins switch from the embryonic epsilon-to the fetal gamma-chains. We investigated the chromatin structure of the human alpha-globin gene cluster in fetal and adult erythroid cells. Our results indicate that DNase I-hypersensitive sites exist at the 5' ends of the alpha 1- and alpha 2-globin genes as well as at several other sites in the cluster in all erythroid cells examined. In addition, early and late fetal liver erythroid cells and adult bone marrow cells contain hypersensitive sites at the 5' end of the zeta gene, and in a purified population of 130-day-old fetal erythroid cells, the entire zeta-to alpha-globin region is sensitive to DNase I digestion. The presence of features of active chromatin in the zeta-globin region in fetal liver and adult bone marrow cells led us to investigate the transcription of zeta in these cells. By nuclear runoff transcription studies, we showed that initiated polymerases are present on the zeta-globin gene in these normal erythroid cells. Immunofluorescence with anti-zeta-globin antibodies also showed that late fetal liver cells contain zeta-globin. These findings demonstrate that expression of the embryonic zeta-globin continues at a low level in normal cells beyond the embryonic to fetal globin switch.

A developmentally stable chromatin structure in the human beta-globin gene cluster.

The DNase I-hypersensitive sites in the human embryonic beta-globin gene region have been mapped in erythroid-enriched fractions of disaggregated fetal livers, in adult nucleated red blood cells, and in fetal brain tissue. Our analysis of a region extending 11 kilobases (kb) 5' of the epsilon-globin gene reveals many minor nuclease-hypersensitive sites and one major site located 6.1 kb upstream of the epsilon-globin gene. All of these hypersensitive sites are erythroid-specific, and the major site is stable throughout erythroid development. As assayed by nuclear runoff transcription, little or no epsilon-globin gene expression is detectable in fetal or adult erythroid cells. Thus, the presence of the major hypersensitive site 5' of the epsilon-globin gene in both fetal and adult erythroid cells demonstrates that this site is not specifically correlated with transcription of the gene or with a particular stage of development. Rather, this site may reflect an early event in erythroid differentiation. In addition, DNase I has been used to probe the overall sensitivity of epsilon-globin chromatin in fetal erythroid cells. Our findings indicate that the epsilon-globin gene as well as the other genes in the beta-globin cluster reside within the chromatin domain that is more DNase I-sensitive than "bulk" chromatin.

Developmental regulation of a cloned adult beta-globin gene in transgenic mice.

At different stages of mammalian development, distinct embryonic, fetal and adult haemoglobins are synthesized in erythroid cells, a process termed haemoglobin switching. The cellular and molecular mechanisms controlling haemoglobin switching have been intensively studied, but remain poorly understood. To study the developmental regulation of globin gene expression, we have produced transgenic mice in which cloned globin genes are present in erythroid cells throughout development. Recently, we reported that adult mice in several transgenic lines carrying a hybrid mouse/human adult beta-globin gene, expressed the gene in a correct tissue-specific manner. This finding raised the question of whether an exogenous globin gene could also be subject to appropriate stage-specific regulation. We report here that the hybrid beta-globin gene, like the endogenous adult beta-globin genes, is inactive in yolk sac-derived embryonic erythroid cells and is expressed for the first time in fetal liver erythroid cells. Our results indicate that a stage-specific pattern of expression can be conferred by cis-acting regulatory elements closely linked to an adult beta-globin gene. They also suggest that the embryonic and adult beta-globin genes in the mouse are activated (or repressed) by distinct trans-acting regulatory factors present in embryonic, fetal and adult erythroid cells.

Activation of a chicken embryonic globin gene in adult erythroid cells by 5-azacytidine and sodium butyrate.

Adult White Leghorn chickens were rendered anemic by injection with 1-acetyl-2-phenylhydrazine and then treated with parenteral 5-azacytidine, and levels of embryonic globin RNA in circulating reticulocytes were measured. A very small but detectable amount of correctly initiated embryonic p-type globin RNA was detected in reticulocytes from birds treated with 5-azacytidine, while none was detected in reticulocytes from those receiving only phenylhydrazine or phenylhydrazine plus 1-beta-D-arabinofuranosylcytosine (cytosine arabinonucleoside). An attempt to increase embryonic globin RNA induction by treatment with parenteral sodium butyrate after 7 days of 5-azacytidine administration resulted in a 5- to 10-fold increase in the level of embryonic globin RNA. However, sodium butyrate did not induce embryonic gene expression when given alone or after treatment with cytosine arabinonucleoside. Sodium butyrate treatment also caused a DNase I-hypersensitive site to be exposed at the 5' end of the rho-globin gene only after 5-azacytidine induced demethylation of several CpG sites in and around the gene. The implications of this model of gene activation in vivo are discussed in the context of multistep gene regulation.

Analysis of rabbit β-like globin gene transcripts during development

We have analyzed the differential expression of a family of β-like globin genes during the development of rabbits, from four days post implantation to one week before birth. The family is composed of four genes, arranged 5′-β4-β3-Ψβ2-β1–3′ on the chromosome; Ψβ2 is an inactive pseudogene. Using the technique of hybrid-arrested translation in vitro, we have identified the embryo-specific globin polypeptides encoded by genes β3 and β4. The β3 and β4 globins are replaced by the adult β1 globin halfway through gestation; this corresponds temporally with the switch in site of erythropoiesis from the embryonic yolk sac to the fetal liver. The decline in production of β3 globin polypeptide precedes the decline in β4 globin. Transcripts from genes β1, β3 and β4 were analyzed at progressive stages of gestation by a blot-hybridization assay and by an S 1 nuclease protection assay. Mature messenger RNA and presumptive precursor RNAs from genes β3 and β4 are synthesized abundantly in embryonic erythroid cells but only at very low levels later in fetal development. Conversely, precursor and mature mRNA from gene β1 are found at very low levels in embryos but are abundant in fetal and adult erythroid cells. The co-ordinate appearance of precursor RNA, mRNA and polypeptide from all three active genes indicates that the primary developmental regulation of this gene family is exerted at the level of transcription. RNA species larger than the expected precursors were observed when the RNA was denatured with formaldehyde but not when methylmercury was the denaturant. These large RNAs are a formaldehyde-generated artifact, possibly a result of cross-linking globin transcripts to ribosomal RNA. We observe no extensive stable transcripts from the 5′ or 3′ flanking regions of these genes.

DNA methylation in chicken alpha-globin gene expression.

We have investigated certain specific methylation sites of the chicken alpha-globin gene cluster in DNA from embryonic and adult erythroid cells as well as from brain and sperm cells. Eight contiguous DNA fragments of the alpha-globin gene cluster were subcloned from a recombinant lambda phage. The subclones were used as probes to map all the Msp I/Hpa II and Hha I sites in the unmethylated cloned DNA and specific sites of methylation in and around the alpha-globin gene cluster in chromosomal DNA. The data show that sperm DNA is totally methylated at these restriction sites in the globin gene region, as is brain DNA, with some exceptions. Interestingly, the methylation status of specific sites 5' to the coding sequences is correlated with expression of the embryonic or adult alpha-globin genes in different stages of erythroid development. Some sites showing partial methylation, however, do not conform to the model that transcribed genes are unmethylated or undermethylated. We also find a well-defined 3.5-kilobase region of DNA 5' to the alpha-globin gene cluster in which all C-C-G-G sites are resistant to Msp I digestion in all tissues. This "Msp block" is presumably caused by 5-MeCpC methylation.

Human hemoglobin switching: insights from studies of erythroid cultures.

Clonal erythroid cultures have been used in the investigation of the cellular mechanisms underlying the switch from fetal to adult Hb formation during ontogeny and the expression of fetal hemoglobin in adult life. It has been shown that adult BFUes have the potential to form F cells and A cells in culture. The cellular segregation in the expression of Hb F within a clone leads to the formation of characteristic bursts displaying a bimorphic F+/F-pattern of Hb F expression. Analysis of distribution of F-expressing and not F-expressing subclones and sectors among bursts of various sizes is compatible with the assumption that progenitor cells exercise options on whether they will or they will not form progeny expressing the Hb F phenotype. Distributions of Hb F expressing subclones among bursts fit the expectations of a stochastic model; probabilities for F expression in the adult erythroid cells may be influenced by the environment in which the cells differentiate and mature. Experimental data from cultures of progenitors from earlier stages of human development indicate that the change in globin expression during ontogeny is controlled at the level of progenitor cells. Observations of Hb F levels produced by single erythroid bursts of the neonate make it unlikely that hemoglobin switching during ontogeny is accomplished through replacement of stem cell lines. The findings in erythroid cultures are compatible with the hypothesis that hemoglobin switching during ontogeny is accomplished through intrinsic or interactive changes in the programs of differentiation of progenitors.

Stochastic expression of fetal hemoglobin in adult erythroid cells.

The expression of fetal hemoglobin, Hb F, in the adult cells is cellularly restricted both in vivo and in culture. Because, in cultures of erythroid progenitors, subclones that express or fail to express Hb F are derived from the same erythroid stem cell, a mechanism must exist whereby Hb F expression segregates in the progeny of erythroid progenitors during their differentiation. We present mathematical analyses of experimental data which suggest that expression of Hb F in human adult erythroid cells occurs on a stochastic basis. We quantified Hb F expression among the subclones of erythroid bursts (clones) in vitro by labeling subclones with fluorescent anti-Hb F antibodies. The observed data were compared with predictions from a stochastic model with the assumption that the expression of Hb F in a subclone occurs with a probability P equal to the frequency of Hb F-expressing subclones in an experiment. There was good fit between the observed and predicted data with respect to: (i) the relative frequencies of monomorphic F+, monomorphic F-, and bimorphic F+/F- bursts, respectively; (ii) the size distributions among F+, F- and F+/F- bursts; and (iii) the proportions of subclones expressing Hb F within bimorphic F+/F- bursts. Given the hypothesis that erythroid progenitors which have an active Hb F program are less differentiated than cells which do not proceed to express Hb F, the stochastic event indicated by our analyses may be the probability that adult erythroid progenitor cells undergo terminal differentiation at an earlier stage than usual.

STRAIN-SPECIFIC APPEARANCE OF A POSSIBLE FETAL α-GLOBIN POLYPEPTIDE CHAIN IN MICE.

DDD mouse embryos at 12-16 days of gestation have a putative 'fetal'α-globin polypeptide chain, detected by its lower electrophoretic mobility than the authentic α-globin chain on acidic urea-Triton gel. When hepatic erythroid cells of DDD embryos were contaminated with yolk sac erythroid cells, two α-bands were visible, but only 'fetal'α seemed to be synthesized, because in embryos of strain ddY which share a common ancestry with DDD, only 'fetal'α was detectable by autoradiography when the hepatic erythroid cells of embryos of more than 15 days of gestation showed an authentic adult α-globin chain other than this 'fetal'α-globin chain. If this 'fetal'α-globin chain is structurally different from the yolk sac α and adult peripheral blood α-globin chains, then switching over of the transcription of α-globin polypeptide chain occurs in hepatic erythroid cells of mid-late DDD embryos. However, it is still possible that this 'fetal'α-globin chain is formed by post-translational modification of the authentic α-globin chain. The α-globin chains of hepatic erythroid cells of C57BL/6 and BALB/c embryos had similar mobilities to those of yolk sac and adult erythroid cells.

Cellular regulation of hemoglobin switching: evidence for inverse relationship between fetal hemoglobin synthesis and degree of maturity of human erythroid cells.

To investigate whether the level of maturity of human erythroid cells influences the expression of the fetal hemoglobin program, we studied the relative production of fetal (Hb F) and adult (Hb A) hemoglobins during the maturation of erythroid clones produced in vitro by adult or neonatal erythroid stem cells. In both the adult and the neonatal cell cultures, clones composed of immature erythroblasts showed a significantly higher Hb F/Hb A ratio compared to the mature clones. Culture conditions enhancing erythroid cell maturity (such as an increase in the level of erythropoietin or culture time) decreased the relative synthesis of Hb F in the maturing erythroid cells. Direct immunofluorescence studies demonstrated earlier production of Hb F compared with Hb A during maturation of adult-origin HbF-synthesizing clones. The findings show that the final expression of Hb F is influenced by the degree of maturity of the terminally differentiated cells and suggest that, in addition to regulation at the level of erythroid stem cells, there is control of Hb F expression during erythroblast maturation. The inverse relationship between Hb F expression and level of cell maturity suggests that a regulatory mechanism operating throughout the process of erythroid stem cell differentiation/erythroblast maturation decreases the potential of Hb F expression as the development of the erythroid cell advances.